Colonization of Rice Roots by a Green Fluorescent Protein-Tagged Isolate of Ustilaginoidea virens

Mebeaselassie Andargie^1*, Luoye Li¹, Aiqing Feng², Jianxiong Li^1*

¹Key Laboratory of South China Agricultural Plant Molecular Analysis and Genetic Improvement, and Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China.
²Plant Protection Institute, Guangdong Academy of Agricultural Sciences, Guangzhou, China.

DOI: 10.4236/ajps.2015.614230                

Abstract

The fungus U. virens is the causal agent of rice false smut disease. The green fluorescent protein (GFP) was used to mark this fungus in order to visualize and analyze the colonization and infection processes in vivo. Using epifluorescence microscopy colonization and infection on rice roots were visualized in vivo. After inoculation for 2 to 15 d, it was observed that the conidia and their germ-tubes had penetrated into epidermis of young roots. The hyphae were found inside the root xylem 18 d after inoculation. Generally, the transformed fungus colonized the rhizosphere, the cortex as well as the vascular tissues with symptoms of root necrosis observed. The results of this work show that U. virens colonize not only rice panicles but also the roots.

Keywords

Endophytes, Plant-Fungal Interactions, Rhizosphere, Root Colonization, Ustilaginoidea virens

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Tanaka, E., Ashizawa, T., Sonoda, R. and Tanaka, C. (2008) Villosiclava virens gen. nov., comb. nov., the Teleomorph of Ustilaginoidea virens, the Causal Agent of Rice False Smut. Mycotaxon, 106, 491-501.
[2]	Singh, S., Pal, V. and Panwar, M. (1992) False Smut of Rice-Its Impact on Yield Components. Crop Research Hisar, 5, 246-248.
[3]	Biswas, A. (2001) Field Reaction of Hybrid Rice Varieties to False Smut and Kernel Smut Disease in West Bengal India. Environment Ecology, 19, 299-230.
[4]	Yang, L.M., Chen, L., Xu, J., Liu, J.C. and Ding, K.J. (2012) Estimation of Yield Loss Caused by Rice False Smut. Journal of Anhui Agricultural University, 39, 474-477.
[5]	Koiso, Y., Li, Y., Iwasaki, S., Hanaoka, K., Kobayashi, T., Sonoda, R., Fujita, Y., Yaegashi, H. and Sato, Z. (1994) Ustiloxins, Antimitotic Cyclic Peptides from False Smut Balls on Rice Panicles Caused by Ustilaginoidea virens. Journal of Antibiotics, 47, 765-773. http://dx.doi.org/10.7164/antibiotics.47.765
[6]	Luduena, R.F., Roach, M.C., Prasad, V., et al. (1994) Interaction of Ustiloxin A with Bovine Brain Tubulin. Biochemical Pharmacology, 47, 1593-1599. http://dx.doi.org/10.1016/0006-2952(94)90537-1
[7]	Schroud, P. and TeBeest, D.O. (2005) Germination and Infection of Rice Roots by Spores of Ustilaginoidea virens. In: Norman, R.J., Meullenet, J.F. and Moldenhauer, K.A.K., Eds., University of Arkansas Agricultural Experiment Station Research Series, 540, 143-151. Fayetteville, Ark.
[8]	Ashizawa, T., Takahashi, M., Arai, M. and Arie, T. (2012) Rice False Smut Pathogen, Ustilaginoidea virens, Invades through Small Gap at the Apex of a Rice Spikelet before Heading. Journal of General Plant Pathology, 78, 255-259. http://dx.doi.org/10.1007/s10327-012-0389-3
[9]	Tang, Y.X., Jin, J., Hu, D.W., Yong, L.M., Xu, Y. and He, L.P. (2013) Elucidation of the Infection Process of Ustilaginoidea virens(Teleomorph: Villosiclava virens) in Rice Spikelets. Plant Pathology, 62, 1-8. http://dx.doi.org/10.1111/j.1365-3059.2012.02629.x
[10]	Lee, F.N. and Gunnell, P.S. (1992) False Smut. In: Webster, R.K. and Gunnell, P.S., Eds., Compendium of Rice Diseases, American Phytopathological Society, St. Paul, Minn. Pg. 28.
[11]	Sukno, S.A., Garcia, V.M., Shaw, B.D. and Thon, M.R. (2008) Root Infection and Systemic Colonization of Maize by Colletotrichum graminicola. Applied and Environmental Microbiology, 74, 823-832. http://dx.doi.org/10.1128/AEM.01165-07
[12]	Olivain, C. and Alabouvette, C. (1997) Colonization of Tomato Root by a Non-Pathogenic Strain of Fusarium oxysporum. New Phytologist, 137, 481-494. http://dx.doi.org/10.1046/j.1469-8137.1997.00855.x
[13]	Olivain, C. and Alabouvette, C. (1999) Process of Tomato Root Colonization by a Pathogenic Strain of Fusarium oxysporum f. sp. lycopersici Discussed in Comparaison to a Non-Pathogenic Strain. New Phytologist, 141, 497-510. http://dx.doi.org/10.1046/j.1469-8137.1999.00365.x
[14]	Olivain, C., Trouvelot, S., Binet, MN., Cordier, C., Pugin, A. and Alabouvette, C. (2003) Colonization of Flax Roots and Early Physiological Responses of Flax Cells Inoculated with Pathogenic and Non-Pathogenic Strains of Fusarium oxysporum. Applied and Environmental Microbiology, 69, 5453-5462. http://dx.doi.org/10.1128/AEM.69.9.5453-5462.2003
[15]	Lagopodi, A.L., Ram, A.F.L., Lamers, G.E.M., Punt, P.J., Van den Hondel, J.J., Lugtenberg, B.J.J. and Bloemberg, G.V. (2002) Novel Aspects of Tomato Root Colonization and Infection by Fusarium oxysporum f. sp. radicis-lyco-persici Revealed by Confocal Laser Scanning Microscopic Analysis Using the Green Fluorescent Protein as a Marker. Molecular Plant-Microbe Interactions, 15, 172-179. http://dx.doi.org/10.1094/MPMI.2002.15.2.172
[16]	Lubeck, M., Knudsen, M.B., Jensen, B., Thrane, U., Janvier, C. and Jensen, D.F. (2002) GUS and GFP Transformation of the Biocontrol Strain Clonostachys rosea IK726 and the Use of These Marker Genes in Ecological Studies. Mycological Research, 106, 815-826. http://dx.doi.org/10.1017/S095375620200607X
[17]	von der Weid, I., Artursson, V., Seldin, L. and Jansson, J.K. (2005) Antifungal and Root Surface Colonization Properties of GFP-Tagged Paenibacillus brasilensis PB177. World Journal of Microbiology and Biotechnology, 21, 1591- 1597. http://dx.doi.org/10.1007/s11274-005-8123-3
[18]	Mansouri, S. and Fakhoury, A.M. (2006) Developing Molecular Tools to Study the Fusarium virguliforme Soybean Interaction. Phytopathology, 96, S72-S73.
[19]	Chalfie, M. and Kain, S. (1998) GFP Green Fluorescent Protein Properties, Applications and Protocols. Wiley-Liss, Inc., New York.
[20]	Tsien, R.Y. (1998) The Green Fluorescent Protein. Annual Review of Biochemistry, 67, 509-544. http://dx.doi.org/10.1146/annurev.biochem.67.1.509
[21]	Rovira, A.D., Newman, E.I., Bowen, H.J. and Campbell, R. (1974) Quantitative Assessment of the Rhizoplane Microflora by Direct Microscopy. Soil Biology and Biochemistry, 6, 211-216. http://dx.doi.org/10.1016/0038-0717(74)90053-4
[22]	Hu, M.L., Luo, L.X., Wang, S., Liu, Y.F. and Li, J.Q. (2013) Infection Processes of Ustilaginoidea virens during Artificial Inoculation of Rice Panicles. European Journal of Plant Pathology, 139, 67-77. http://dx.doi.org/10.1007/s10658-013-0364-7
[23]	Mebeaselassie, A., Li, L.Y., Feng, A.Q., Zhu, X.Y. and Li, J.X. (2015) Development of a GFP-Expressing Ustilaginoidea virens Strain to Study Fungal Invasion and Colonization in Rice Spikelets. South African Journal of Botany, 97, 16-24. http://dx.doi.org/10.1016/j.sajb.2014.11.013
[24]	Parsa, S., Ortiz, V. and Vega, F.E. (2013) Establishing Fungal Entomopathogens as Endophytes: Towards Endophytic Biological Control. Journal of Visualized Experiments, 74, e50360. http://dx.doi.org/10.3791/50360
[25]	Smith, S.E. and Read, D.J. (1997) Mycorrhizal Symbiosis. 2nd Edition, Academic Press, San Diego.
[26]	Harrison, M.J. (1999) Molecular and Cellular Aspects of the Arbuscular Mycorrhizal Symbiosis. Annual Review of Plant Physiology and Plant Molecular Biology, 50, 361-389. http://dx.doi.org/10.1146/annurev.arplant.50.1.361
[27]	Parniske, M. (2000) Intracellular Accommodation of Microbes by Plants: A Common Development Program for Symbiosis and Disease? Current Opinion in Plant Biology, 3, 320-328. http://dx.doi.org/10.1016/S1369-5266(00)00088-1
[28]	Strack, D., Fester, T., Hause, B., Schliemann, W. and Walter, M.H. (2003) Arbuscular mycorrhiza: Biological, Chemical, and Molecular Aspects. Journal of Chemical Ecology, 29, 1955-1979. http://dx.doi.org/10.1023/A:1025695032113
[29]	Lorang, J.M., Tuori, R.P., Martinez J.P., Sawyer, T.L., Redman, R.S., Rollins, J.A., et al. (2001) Green Fluorescent Protein Is Lighting up Fungal Biology. Applied and Environmental Microbiology, 67, 1987-1994. http://dx.doi.org/10.1128/AEM.67.5.1987-1994.2001
[30]	Oren, L., Ezrati, S., Cohen, D. and Sharon, A. (2003) Early Events in the Fusarium verticillioides-Maize Interaction Characterized by Using a Green Fluorescent Protein Expressing Transgenic Isolate. Applied and Environmental Microbiology, 69, 1695-1701. http://dx.doi.org/10.1128/AEM.69.3.1695-1701.2003
[31]	Visser, M., Gordon, T.R., Wingfield, B.D., Wingfield, M.J. and Viljoen, A. (2004) Transformation of Fusarium oxysporum f. sp. cubense, Causal Agent of Fusarium Wilt of Banana, with the Green Fluorescent Protein(GFP) Gene. Australasian Plant Pathology, 33, 69-75. http://dx.doi.org/10.1071/AP03084
[32]	Li, C.Y., Chen, S., Zuo, C.W., Sun, Q.M., Ye, Q., Yi, G.J. and Huang, B.Z. (2011) The Use of GFP Transformed Isolates to Study Infection of Banana with Fusarium oxysporum f. sp. cubense Race 4. European Journal of Plant Pathology, 131, 327-340. http://dx.doi.org/10.1007/s10658-011-9811-5
[33]	Damm, U., Brune, A. and Mendgen, K. (2003) In Vivo Observation of Conidial Germination of the Oxi-Anoxic Interface and Infection of Submerged Reed Roots by Microdochium bolleyi. FEMS Microbiology Ecology, 45, 293-299. http://dx.doi.org/10.1016/S0168-6496(03)00161-2
[34]	Gao, K.X. and Mendgen, K. (2006) Seed-Transmitted Beneficial Endophytic Stagonospora sp. Can Penetrate the Walls of the Root Epidermis, but Does Not Proliferate in the Cortex, of Phragmites australis. Canadian Journal of Botany- Revue Canadienne de Botanique, 84, 981-988.
[35]	Zvirin, T., Herman, R., Brotman, Y., Denisov, Y., Belausov, E., Freeman, S., et al. (2010) Differential Colonization and Defence Responses of and Susceptible Melon Lines Infected by Fusarium oxysporum Race 1.2. Plant Pathology, 59, 576-585. http://dx.doi.org/10.1111/j.1365-3059.2009.02225.x
[36]	An, Q.L., Yang, X.J., Dong, Y.M., Feng, L.J., Kuang, B.J. and Li, J.D. (2001) Using Confocal Laser Scanning Microscope to Visualize the Infection of Rice Roots by GFP-Labelled Klebsiella oxytoca SA2, an Endophytic Diazotroph. Acta Botanica Sinica, 43, 558-564.
[37]	Eynck, C., Koopmann, B., Grunewaldt-Stoecker, G., Karlovsky, P. and von Tiedemann, A. (2007) Differential Interactions of Verticillium longisporum and V. dahliae with Brassica napus Detected with Molecular and Histological Techniques. European Journal of Plant Pathology, 118, 259-274. http://dx.doi.org/10.1007/s10658-007-9144-6
[38]	Abdellatif, L., Bouzid, S. and Vujanovic, V. (2007) New Plant Growth Promoting Fungal Endophytes Reprogram Wheat and Provide Disease Resistance. Plant Canada—Growth for the Future (Proceedings), Saskatoon, SK, Canada, 137-139.